(a) Field of the Invention
The present invention relates to a hyaluronic acid producing microorganism strain and a method for purifying hyaluronic acid, and particularly to Streptococcus sp. KL0188 and a method for purifying hyaluronic acid using an aromatic adsorption resin and an active carbon.
(b) Description of the Related Art
Hyaluronic acid, which is a colorless highly viscous polysaccharide having a molecular weight of 50,000 to 13,000,000 Da, has repeat units of glucuronic acid and N-acetyl glucosamine that are alternately bonded as (1-3) and (1-4). Hyaluronic acid has a moisturizing effect, an excellent lubricating effect against physical friction, and it offers excellent protection effects against invasion of bacteria, etc. Hyaluronic acid is widely used as a cosmetic additive, a treating agent for arthritis, a supplementary agent for ophthalmic operations, an adhesion inhibitor after surgical operations, etc. A lot of hyaluronic acid is contained in cow eyeballs, cock combs, buffer tissue of animals, placentas, cancer cells, skin, etc.
Hyaluronic acid can be extracted from the above-mentioned bio-tissues (U.S. Pat. No. 4,141,973 and U.S. Pat. No. 4,303,676), or it can be collected as a fermentation product by fermenting microorganisms. However, hyaluronic acid obtained by extraction contains impurities such as chondroitin sulfate, glycosamino glycansulfate, etc. hence a complicated purification process is required in order to remove these impurities causing a high production cost. However, according to the production method of hyaluronic acid using microorganisms, production cost is comparatively low, and high molecular weight hyaluronic acid can be obtained by a relatively simple method (Japanese Patent Laid-Open publication No. 58-056692, U.S. Patent 86-00066).
Microorganisms used for the production of hyaluronic acid include Streptococcus pyogenes, Streptococcus faecalis, Streptococcus dysgalactiae, Streptococcus zooepidemicus, Streptococcus equi, Streptococcus equisimilis, etc. According to Bergy's manual, these pertain to Lancefield's serological group A or C. Such microorganisms are hemolytic Streptococcus, and they are reported to have beta-hemolytic functions.
Since hyaluronic acids that are produced using Streptococcus sp. microorganisms (Japanese Patent Laid-Open Publication No. 58-566922, U.S. Patent Laid-Open Publication No. 60-500997, Korean Patent Registration Publication No. 10-250573, and Korean Patent Laid-Open Publication No. 10-250573) have relatively low average molecular weights of 300,000 to 3,500,000 Da, it is difficult to use them as a medicinal treating agent or supplementary agent, and they have insufficient moisturizing power for cosmetics. In addition, U.S. Pat. No. 6,090,596 describes a method for producing high molecular weight hyaluronic acid with molecular weight of 6,300,000 to 9,500,000 Da, but productivity of hyaluronic acid is 0.35 g per L of culture solution, which is very low.
Known methods for separating and purifying hyaluronic acid using microorganisms are as follows:
U.S. Pat. No. 4,157,296 discloses a method for purifying hyaluronic acid by treating a culture solution of Streptococcus pyogenes with trichloro acetic acid to remove strains, and then precipitating it using an organic solvent. However, since the precipitation method using an organic solvent requires numerous repetitive operations, it has a comparatively high cost and consumes substantial time.
U.S. Pat. No. 4,782,046 describes a purification process of introducing 0.01% anionic surfactant of lauryl sulfate into a culture solution of Streptococcus equi to separate hyaluronic acid attached to cell walls, and then introducing a non-ionic surfactant of hexadecyltrimethyl ammonium bromide to form a hyaluronic acid precipitate, and precipitating it with alcohol.
U.S. Pat. No. 4,784,990 describes a purification process of adding ethanol to a culture solution of Streptococcus zooepidemicus to separate hyaluronic acid from microorganisms, and then precipitating it with cetyl pyridinium chloride.
Japanese Patent Laid-Open Publication No. 63-012293 describes a method for removing both low molecular weight hyaluronic acid with a molecular weight of 1,500,000 Da or less and exothermic material by treating a hyaluronic acid-containing solution with a macroreticular anion exchange resin (Dianion HPA-25, HPA-75, IRA-900, IRA-904).
Japanese Patent Laid-Open Publication No. 13-131503 describes a method for purifying hyaluronic acid by treating a hyaluronic acid-containing solution with alumina or silica gel, etc. to remove exothermic material, proteins, nucleic acid, metal impurities, etc., and precipitating it with an organic solvent.
Japanese Patent Laid-Open Publication No. 06-199656 describes a method for purifying hyaluronic acid by passing a hyaluronic acid-containing solution through a membrane filter charged on a solution of pH 6 to 10 to remove exothermic material, and precipitating it with alcohol.
Korean Patent Laid-Open Publication No. 1994-2478 describes a method for purifying hyaluronic acid by adding iron aluminate powders to a hyaluronic acid producing strain culture solution.
In addition, Korean Patent Laid-Open Publication No. 1997-42603 describes a method for purifying hyaluronic acid by treating a hyaluronic acid-containing solution with a hydrophobic polymer (polyethylene, polypropylene, or polystyrene), and then adding active alumina, and precipitating it with alcohol.
However, the above-mentioned methods involve complicated treating processes, which increase production cost, and it is difficult to completely remove exothermic material, proteins, nucleic acid, etc.